Project Summary/Abstract The main aims of this multiple-PI proposal are to evaluate the safety and eficacy of, and the host immune response to, the next generation of recombinant adeno-associated virus (AAV) vectors that we have developed, in small and large animal models of human liver diseases in general, and hemophilia in particular. AAV vectors have gained attention as an alternative to the more commonly used retrovirus and adenovirus vectors, and are in use in Phase I/II clinical trials for gene therapy of a number of diseases. However, relatively large vector doses are needed to achieve therapeutic benefits. Large vector doses also trigger an immune response as a significant fraction of the vectors fails to traffic efficiently to the nucleus, and is targeted for degradation by the host cell proteasome machinery. Our recent studies have yielded insights into key steps in intracellular trafficking of AAV, and led to the development of novel AAV vectors that are capable of high-efficiency transduction at lower doses. We will test the following hypotheses: a. Combination of specific tyrosine mutations in AAV2 capsids will further reduce the vector dose needed for high-efficiency transduction, and corresponding mutations in tyrosine residues in AAV8 and AAV5 serotype vectors will lead high-efficiency transduction of murine and canine hepatocytes. b. Novel Baculovirus system-produced rAAV vectors, characterized by higher VP1 capsid protein stoichiometric content, will exhibit superior transduction properties in target tissues. c. Tyrosine-mutant AAV vectors will elicit a reduced host cell immune response, and provide therapeutic benefits at lower doses. The following three Specific Aims will be pursued: 1. Development of AAV2 vectors containing multiple tyrosine-mutations, elucidation of the underlying mechanism of transduction by the most efficient vector in vitro and in vivo, and comparative analysis with AAV8 and AAV5 vectors. 2. Development of the next generation of Sf9-based stable cell lines for the production of highly infectious rAAV of alternative serotypes. 3. Treatment of murine and canine hemophilia B with optimal tyrosine-mutant AAV2, AAV8, and AAV5 serotype vectors and evaluation of immune responses to vector and coagulation factor IX transgene product. The knowledge gained from these studies will not only shed light on the AAV-host cell interactions, but will also be applicable in further improvements in recombinant AAV vectors for their potential use in gene therapy of human liver diseases in general, and hemophilia in particular. PUBLIC HEALTH RELEVANCE: Project Narrative The main aim of this proposal is to develop the next generation of vectors with which a therapeutic gene can be safely delivered to patients with a bleeding disorder called hemophilia B. These vectors are derived from a virus that causes no known disease, and is therefore, expected to be safer. The development of such a vector for the potential treatment and cure of hemophilia therefore has relevance to public health.